Assessing the efficiency of high-resolution melting, conventional PCR, and capillary electrophoresis in SSR marker-based genotyping of carob populations

Ceratonia siliqua is a nutritionally and economically important Mediterranean tree species whose natural stands in Morocco are increasingly threatened by habitat degradation and genetic erosion. However, the spatial organisation of genetic diversity across its natural range in Morocco remains insufficiently resolved. To support conservation and germplasm management initiatives, interpopulation genetic differentiation among Moroccan carobs populations was assessed using a pooled DNA sample and SSR markers analysed through three genotyping workflows: high-resolution melting (HRM), conventional PCR, and capillary electrophoresis. Genetic relationships among populations were evaluated using Neighbour-Joining (NJ) analysis, principal component analysis (PCA), and K-means clustering. Among the evaluated approaches, HRM provided the highest discriminatory resolution and generated the most informative polymorphism profiles across loci. All analytical frameworks consistently revealed substantial interpopulation differentiation and broad geographic grouping patterns. Southern populations formed genetically cohesive groups, whereas northern and central populations displayed more complex patterns of genetic similarity. K-means clustering supported the presence of four major population groups, while PCA identified a subset of highly discriminative SSR loci contributing to regional differentiation. Overall, the results reveal a genetically diverse and spatially differentiated Moroccan carob germplasm and demonstrate that SSR-HRM is a robust and cost-effective approach for population-level genetic screening. These findings provide useful genetic resources for future conservation, germplasm management, and breeding programmes in Moroccan carob.